Abstract
A protein called DNA Polymerases is capable of creating new DNA strands by incorporating the monomeric units of DNA called nucleotides opposite the single stranded parent DNA in a Watson Crick manner (A:T and G:C). DNA polymerases are therefore centrally involved in the replication of DNA. In order to carry out this crucial function, these replicative DNA polymerase molecules exhibit extremely high accuracy and processivity (measure of the no. of nucleotides added by a polymerase per association with a DNA). The presence of damaged nucleotides in the genome (DNA code for any organism) is highly inhibitory to the activity of these DNA Polymerases. DNA can be damaged by a variety of external (radiation, chemicals) and internal (free radicals, reactive intermediates) agents. Replicative DNA Polymerases are not capable of stabilizing the damaged nucleotide and the correct incoming nucleotide in their active sites which leads to the stalling of the replication machinery. To overcome this predicament it is seen that all organisms have specialized DNA Polymerases grouped under the Y-family of DNA Polymerases. The members of this family usually exhibit low-fidelity (ability to pair in Watson Crick manner) and low-processivity and are able to incorporate nucleotides opposite various kinds of damaged nucleotides. It is believed that once these molecules help the replication machinery cross the damage they are replaced by normal replicative DNA Polymerases. In addition, it has also been suggested that the low-fidelity of these molecules is exploited to generate adaptive mutations that can relieve selection pressure arising from adverse environmental conditions. This might be especially true in case of prokaryotes (bacterial organisms) where the expression of these molecules is controlled by transcriptional mechanisms that deal with stress. We have carried out structural and functional studies on one of theY-family protein in Mycobacterium smegmatis (bacterial organism similar toTuberculosis and non pathological), MsDpo4. The aim is to provide atopological and chemical description of MsDpo4 in order to understand the relation between structure and function in case of Y- Family DNA Polymerases in prokaryotes. We have seen that this protein prefers Watson-Crick mode of base pairing and it is also capable of promoting mismatches. Steady-state kinetic analysis shows that this protein exhibits significant ability to promote G:T and T:G mismatches and thus has the biochemical capacity to participate in adaptive mutagenesis. MsDpo4 is also capable of carrying out synthesis across the damaged DNA. The structure of MsDpo4 has been determined to a resolution of 2.6 Ã…. The structure suggests that one of the domain of this protein exhibits conformational heterogeneity. The possible implications of this observation will be discussed.
